The present invention relates generally to an apparatus for reading flat, two-dimensional gel electrophoretograms, and more particularly, to the use of a proportional chamber radiation detector having two-dimensional particle location capability to electornically read a two-dimensional gel electrophoretogram which has been labeled with .beta.-ray-emitting compounds.
Two-dimensional electrophoresis has been increasingly used in a wide variety of studies involving the detection and analysis of proteins from complex biological systems. The technique holds promise for determining, at a molecular level, how living cells work. See, e.g., "The Proteins of Oncogenes," by Tony Hunter, Scientific American 251, 70, (1984), and, in particular, the figure on page 76 thereof for a brief description of gel electrophoretic analysis of proteins. In the technique of two-dimensional electrophoresis, the proteins are separated according to isoelectric pH point by isoelectric focusing in one dimension, and according to molecular weighty by sodium dodecyl sulfate in the second dimension at right angles to the first. Since these two parameters are uncorrelated, it is possible to obtain a quantitative distribution of protein spots over a two-dimensional gel. The location and intensity of each spot is generally made visible by autoradiography when it is desired to evaluate the entire pattern. Often, large numbers of proteins are present, and the amount of potential information contained within a single two-dimensional gel is large. Although progress has been made in automating the procedures involved and in the computer analysis of the patterns obtained, densitometry measurements of a autoradiograph suffer from a number of problems inherent in the photographic process. First, the photographic emulsion is relatively insensitive to .beta.-particles from .sup.14 C and .sup.35 S, two commonly used radioisotopes for labeling proteins. A deposition of approximately 20,000 disintegrations per mm.sup.2 is needed to produce a minimally detectable image. In practice, a gel containing about 10.sup.6 dpm of a total protein preparation will require an exposure time of about one week. Moreover, protein concentration present in a typical cell under investigation have been estimated to range over six orders of magnitude. Such a dynamic range far exceeds the usable optical density range of photographic film, and requires that a graded series of exposures be obtained for each gel. Exposure times of three or four weeks are not uncommon where species occurring in low abundance are of interest, and the resulting exposures are often unreadable over large areas due to the effects of overexposure from more abundant species. This results in the loss of information for some of the proteins present in smaller quantities within these overexposed areas. Although the total number of proteins estimated to exists in a human cell is about 50,000, only 1 to 3% have been studies in reasonable detai. In view of the importance of proteins in life processes and their potential role in the diagnosis and treatment of diseases, an instrument that is capable of expeditious protein anaylsis with high sensitivity would have significant influence on the development of this important field of investigation.
In "Simple Electronic Apparatus for the Analysis of Radioactively Labeled Gel Electrophoretograms," U.S. Pat. No. 4,311,908, issued to Konstantin Goulianos, Karen K. Smith, and Sebastian N. White on Jan. 19, 1982, the inventors describe a high resolution position-sensitive radiation detector for analyzing the radiation emanating from a .beta.-ray-labeled gel electrophoretogram. A coil used for ionization detectio, also seves as a delay line for determining the location of ionization produced in the vicinity of an anode wire along this anode wire. The geometrical error in determining the point at which a particular .beta.-ray emerges from the gel source is reduced by making the distance between the gel and the anode wire very short and by using the well-known "magic" gas mixture which maximized the number of ion pairs created by the emerging .beta.-rays as they pass through the ionization detection apparatus. In this manner the inventors state that a uniform resolution of about .+-.0.5 MM at the surface of the gel can be achieved by their invention. However, the location of the source of the .beta.-ray in the gel is determined in only one dimension according to the teachings thereof.
In "High Accuracy, Bidimensional Read-Out of Proportional Chambers with Short Resolution Times," by A. Breskin, G. charpak, C. Demierra, S. Majewski, A. Policarpo, F. Sauli, and J. C. Santiard, 143 Nucl. Instrum. Meth. 29 (1977), the authors disclose the use of a multiwire proportional chamber having orthogonal cathode strips to determine the two-dimensional charge centroid of an avalanche produced by the interaction of .beta.-rays with a target gas in the region of a nearby anode which induces positive charges thereon. For a ionizing beam of normal incidence to the plane of the cathodes, a resoltuion of about 200 .mu.m is reported. The resoltuion of incident ionizing particles at higher angles is improved by electronically decreasing the observation time of the generated signal. However, since the device described therein is used to accurately determine particle tracks where few particles are emerging from the same location, no concern is given to narrowing the distribution of incident particles. That is, in the situation where large numbers of particles are isotropically emitted from a single, albeit somewhat diffuse source, such as a labeled protein on a gel, the signals generated therefrom in the multiwire proportional chamber will in general be impossible to analyze unless the divergence of the particles is reduced so that their origin can be more clearly identified.
P. G. Seiler, R. Dietlicher, G. Wemmers, M. Salzmann, and A. Moline in "Two-Dimensional Measurement of Pion-Induced Beta Activity in Extended Foils," 27 Phys. Med. Biol. 709 (1982), describe a procedure for improving the spatial resolution of the two-dimensional measurement of the initial location of .beta.-rays in a foil using multiwire proportional chamber technology. Therein, the authors show that the application of a strong magnetic field perpendicular to the foil under investigation and simultaneously passing through the two proportional chambers utilized therefor in a perpendicular manner relative to the planar multiwire electrodes upon which the two dimensional measurements are made, dramatically improves the resoltuion of the measurements. The .beta.-rays are emitted isotropically from the foils and, due to the finite thickness of the proportional chambers, the .beta.-rays are measured at some distance from their origin. These two factors contribute to the smearing of the resulting electronically derived distribution and correspondingly poor ultimate resolution. The .beta.-rays are constrained to move in a helical path with decreasing radius under the influence of the magnetic field as they lose energy by collisions with the detection gas utilized in the proportional chamber, thereby more accurately preserving the coordinates of their origin within the film. The positions of the decaying nuclei can be determined with an accuracy of about 2.5 mm according to the authors.
Seiler et al., supra, also describe the compensation for cosmic radiation. To achieve this goal, two proportional chambers are employed; one on each side of the film under investigation, and parallel thereto. Thus cosmic rays entering one proportional chamber and causing a signal to be generated therein will most likely enter the second proportional chamber and cause a signal therein a short time thereafter. Such coincident pairs of signals are rejected by the logic circuitry employed. Similarly, pairs of near coincident .beta.-ray signals occurring in the opposingly disposed proportional chambers will be rejected. However, one advantage of the Seiler et al. technique is that one may obtain the absolute activity and a profile of the radioactive sources within the thin foil under investigation twice as efficientlysince the .beta.-rays are emitted and detected both above and below the film.
Accordingly, it is an object of the present invention to provide an apparatus and method for accurately reading planar gel electrophoretograms which ahve been labeled with .beta.-ray-emitting compounds.
Another object of our invention is to provide an apparatus and method for permitting measurements to be made on planar gel electrophoretograms which have been labeled with .beta.-ray-emitting compounds in the presence of unwanted stray cosmic ray events.
Yet another object of the present invention is to provide an apparatus and method for quantitatively and linearly reading planar gel electrophoretograms which have been labeled with .beta.-ray-emitting compounds having a wide range of concentrations thereof.
Additional objects, advantagegs and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.